"I Shall Live and Not Die": Using Monologues Based on the Experiences of Older African Americans Living With HIV to Address HIV-Related Stigma Among African Americans in Louisville, Kentucky.

Remediating racial/ethnic HIV inequities necessitates addressing HIV-related stigma. Arts- and media-based approaches demonstrate potential for effective knowledge translation and HIV-related stigma reduction. This study employs 5 monologues portraying lived experiences of older African Americans living with HIV to do this. Monologues were developed on the basis of qualitative research, actors performed them for live and online audiences, and surveys were distributed to gauge their potential for raising awareness about HIV-related stressors, reducing HIV-related stigma, and entertainment value. Monologues may also foster HIV testing. More scholarship should integrate arts-based knowledge translation with HIV education. Future efforts should focus on scaling this approach.

Variation in the Early Host-Pathogen Interaction of Bovine Macrophages with Divergent Mycobacterium bovis Strains in the United Kingdom.

Bovine tuberculosis has been an escalating animal health issue in the United Kingdom since the 1980s, even though control policies have been in place for over 60 years. The importance of the genetics of the etiological agent, Mycobacterium bovis, in the reemergence of the disease has been largely overlooked. We compared the interaction between bovine monocyte-derived macrophages (bMDM) and two M. bovis strains, AF2122/97 and G18, representing distinct genotypes currently circulating in the United Kingdom. These M. bovis strains exhibited differences in survival and growth in bMDM. Although uptake was similar, the number of viable intracellular AF2122/97 organisms increased rapidly, while G18 growth was constrained for the first 24 h. AF2122/97 infection induced a greater transcriptional response by bMDM than G18 infection with respect to the number of differentially expressed genes and the fold changes measured. AF2122/97 infection induced more bMDM cell death, with characteristics of necrosis and apoptosis, more inflammasome activation, and a greater type I interferon response than G18. In conclusion, the two investigated M. bovis strains interact in significantly different ways with the host macrophage. In contrast to the relatively silent infection by G18, AF2122/97 induces greater signaling to attract other immune cells and induces host cell death, which may promote secondary infections of naive macrophages. These differences may affect early events in the host-pathogen interaction, including granuloma development, which could in turn alter the progression of the disease. Therefore, the potential involvement of M. bovis genotypes in the reemergence of bovine tuberculosis in the United Kingdom warrants further investigation.

Chicken anaemia virus evades host immune responses in transformed lymphocytes.

Chicken anaemia virus (CAV) is a lymphotropic virus that causes anaemia and immunosuppression in chickens. Previously, we proposed that CAV evades host antiviral responses in vivo by disrupting T-cell signalling, but the precise cellular targets and modes of action remain elusive. In this study, we examined gene expression in Marek's disease virus-transformed chicken T-cell line MSB-1 after infection with CAV using both a custom 5K immune-focused microarray and quantitative real-time PCR at 24, 48 and 72 h post-infection. The data demonstrate an intricate equilibrium between CAV and the host gene expression, displaying subtle but significant modulation of transcripts involved in the T-cell, inflammation and NF-κB signalling cascades. CAV efficiently blocked the induction of type-I interferons and interferon-stimulated genes at 72 h. The cell expression pattern implies that CAV subverts host antiviral responses and that the transformed environment of MSB-1 cells offers an opportunistic advantage for virus growth.

The MG-RAST metagenomics database and portal in 2015.

MG-RAST (http://metagenomics.anl.gov) is an open-submission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. The system currently hosts over 200,000 datasets and is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. To show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.

Genomic regions underlying susceptibility to bovine tuberculosis in Holstein-Friesian cattle.

BACKGROUND: The significant social and economic loss as a result of bovine tuberculosis (bTB) presents a continuous challenge to cattle industries in the UK and worldwide. However, host genetic variation in cattle susceptibility to bTB provides an opportunity to select for resistant animals and further understand the genetic mechanisms underlying disease dynamics. METHODS: The present study identified genomic regions associated with susceptibility to bTB using genome-wide association (GWA), regional heritability mapping (RHM) and chromosome association approaches. Phenotypes comprised de-regressed estimated breeding values of 804 Holstein-Friesian sires and pertained to three bTB indicator traits: i) positive reactors to the skin test with positive post-mortem examination results (phenotype 1); ii) positive reactors to the skin test regardless of post-mortem examination results (phenotype 2) and iii) as in (ii) plus non-reactors and inconclusive reactors to the skin tests with positive post-mortem examination results (phenotype 3). Genotypes based on the 50 K SNP DNA array were available and a total of 34,874 SNPs remained per animal after quality control. RESULTS: The estimated polygenic heritability for susceptibility to bTB was 0.26, 0.37 and 0.34 for phenotypes 1, 2 and 3, respectively. GWA analysis identified a putative SNP on Bos taurus autosomes (BTA) 2 associated with phenotype 1, and another on BTA 23 associated with phenotype 2. Genomic regions encompassing these SNPs were found to harbour potentially relevant annotated genes. RHM confirmed the effect of these genomic regions and identified new regions on BTA 18 for phenotype 1 and BTA 3 for phenotypes 2 and 3. Heritabilities of the genomic regions ranged between 0.05 and 0.08 across the three phenotypes. Chromosome association analysis indicated a major role of BTA 23 on susceptibility to bTB. CONCLUSION: Genomic regions and candidate genes identified in the present study provide an opportunity to further understand pathways critical to cattle susceptibility to bTB and enhance genetic improvement programmes aiming at controlling and eradicating the disease.

Enhancing the toolbox to study IL-17A in cattle and sheep.

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

A RESTful API for accessing microbial community data for MG-RAST.

Metagenomic sequencing has produced significant amounts of data in recent years. For example, as of summer 2013, MG-RAST has been used to annotate over 110,000 data sets totaling over 43 Terabases. With metagenomic sequencing finding even wider adoption in the scientific community, the existing web-based analysis tools and infrastructure in MG-RAST provide limited capability for data retrieval and analysis, such as comparative analysis between multiple data sets. Moreover, although the system provides many analysis tools, it is not comprehensive. By opening MG-RAST up via a web services API (application programmers interface) we have greatly expanded access to MG-RAST data, as well as provided a mechanism for the use of third-party analysis tools with MG-RAST data. This RESTful API makes all data and data objects created by the MG-RAST pipeline accessible as JSON objects. As part of the DOE Systems Biology Knowledgebase project (KBase, http://kbase.us) we have implemented a web services API for MG-RAST. This API complements the existing MG-RAST web interface and constitutes the basis of KBase's microbial community capabilities. In addition, the API exposes a comprehensive collection of data to programmers. This API, which uses a RESTful (Representational State Transfer) implementation, is compatible with most programming environments and should be easy to use for end users and third parties. It provides comprehensive access to sequence data, quality control results, annotations, and many other data types. Where feasible, we have used standards to expose data and metadata. Code examples are provided in a number of languages both to show the versatility of the API and to provide a starting point for users. We present an API that exposes the data in MG-RAST for consumption by our users, greatly enhancing the utility of the MG-RAST service.

Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of gastroenteritis in cattle and humans. Dendritic cells (DC) and macrophages (Mø) are major players in early immunity to Salmonella, and their response could influence the course of infection. Therefore, the global transcriptional response of bovine monocyte-derived DC and Mø to stimulation with live and inactivated S. Typhimurium was compared. Both cell types mount a major response 2 h post infection, with a core common response conserved across cell-type and stimuli. However, three of the most affected pathways; inflammatory response, regulation of transcription and regulation of programmed cell death, exhibited cell-type and stimuli-specific differences. The expression of a subset of genes associated with these pathways was investigated further. The inflammatory response was greater in Mø than DC, in the number of genes and the enhanced expression of common genes, e.g., interleukin (IL) 1B and IL6, while the opposite pattern was observed with interferon gamma. Furthermore, a large proportion of the investigated genes exhibited stimuli-specific differential expression, e.g., Mediterranean fever. Two-thirds of the investigated transcription factors were significantly differentially expressed in response to live and inactivated Salmonella. Therefore the transcriptional responses of bovine DC and Mø during early S. Typhimurium infection are similar but distinct, potentially due to the overall function of these cell-types. The differences in response of the host cell will influence down-stream events, thus impacting on the subsequent immune response generated during the course of the infection.

The level of H2O2 type oxidative stress regulates virulence of Theileria-transformed leukocytes.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1ß, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella.

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

The complete genome sequence for putative H2- and S-oxidizer Candidatus Sulfuricurvum sp., assembled de novo from an aquifer-derived metagenome.

We reconstructed the complete 2.4 Mb-long genome of a previously uncultivated epsilonproteobacterium, Candidatus Sulfuricurvum sp. RIFRC-1, via assembly of short-read shotgun metagenomic data using a complexity reduction approach. Genome-based comparisons indicate the bacterium is a novel species within the Sulfuricurvum genus, which contains one cultivated representative, S. kujiense. Divergence between the species appears due in part to extensive genomic rearrangements, gene loss and chromosomal versus plasmid encoding of certain (respiratory) genes by RIFRC-1. Deoxyribonucleic acid for the genome was obtained from terrestrial aquifer sediment, in which RIFRC-1 comprised ∼ 47% of the bacterial community. Genomic evidence suggests RIFRC-1 is a chemolithoautotrophic diazotroph capable of deriving energy for growth by microaerobic or nitrate-/nitric oxide-dependent oxidation of S°, sulfide or sulfite or H2oxidation. Carbon may be fixed via the reductive tricarboxylic acid cycle. Consistent with these physiological attributes, the local aquifer was microoxic with small concentrations of available nitrate, small but elevated concentrations of reduced sulfur and NH(4)(+) /NH3-limited. Additionally, various mechanisms for heavy metal and metalloid tolerance and virulence point to a lifestyle well-adapted for metal(loid)-rich environments and a shared evolutionary past with pathogenic Epsilonproteobacteria. Results expand upon recent findings highlighting the potential importance of sulfur and hydrogen metabolism in the terrestrial subsurface.

Immature dendritic cell-targeting mRNA vaccine expressing PfCSP enhances protective immune responses against Plasmodium liver infection.

Resurgence in malaria has been noted in 2022 with 249 million clinical cases resulting in 608,000 deaths, mostly in children under five. Two vaccines, RTS, S, and more recently R21, targeting the circumsporozoite protein (CSP) are recommended by the WHO but are not yet widely available. Strong humoral responses to neutralize sporozoites before they can infect the hepatocytes are important for vaccine-mediated protection. Suboptimal protection conferred by these first-generation vaccines highlight the need for approaches to improve vaccine-induced immune responses. With the recent success of mRNA-LNP vaccines against COVID-19, there is growing interest in leveraging this approach to enhance malaria vaccines. Here, we present the development of a novel chemokine fusion mRNA vaccine aimed at boosting immune responses to PfCSP by targeting the immunogen to immature dendritic cells (iDC). Vaccination of mice with mRNA encoding full-length CSP fused to macrophage inflammatory protein 3 alpha (MIP3α) encapsulated within lipid nanoparticles (LNP) elicited robust CD4+ T cell responses and enhanced antibody titers against NANP repeat epitopes compared to a conventional CSP mRNA-LNP vaccine. Importantly, the CSP-MIP3α fusion vaccine provided significantly greater protection against liver infection upon challenge with P. berghei PfCSP transgenic sporozoites. This enhanced protection was associated with multifunctional CD4+ T cells levels and anti-NANP repeat titers. This study highlights the potential to augment immune responses to PfCSP through iDC targeting and bolster protection against malaria liver infection.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared. RESULTS: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows. CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1ß was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Community arts engagement supports perceptions of personal growth in older adults.

PURPOSE: The effects of arts engagement on older adults have been well-documented. However, the ways older adults overcome common situational and dispositional barriers to enhance personal growth and well-being are less known. METHODS: Fifty-six community dwelling older adults (71.3 ± 4.6 years) took part in dance, music, or a control workshop two times/week for ten weeks. Participants' personal growth was examined through focus groups and surveys in this mixed-methods study. RESULTS: Focus group and survey results revealed participants experienced personal growth through engaging in the dance and music arms of the experiment. Participants, especially those in arts workshops, described personal growth experiences aligning with four themes: increased social connections, developed new skills, utilized a growth mindset, and used creativity to overcome situational and dispositional barriers to participation. The barriers included musculoskeletal challenges, hearing impairments, and difficulty retaining new information. CONCLUSIONS: The study yielded high adherence and retention rates, and participants reported increased engagement within their communities. Our observations provide avenues for future practitioners and facilitators to create programming that empowers older adults and utilizes participants' ongoing feedback to support access, inclusion, and sense of community.

The M5nr: a novel non-redundant database containing protein sequences and annotations from multiple sources and associated tools.

BACKGROUND: Computing of sequence similarity results is becoming a limiting factor in metagenome analysis. Sequence similarity search results encoded in an open, exchangeable format have the potential to limit the needs for computational reanalysis of these data sets. A prerequisite for sharing of similarity results is a common reference. DESCRIPTION: We introduce a mechanism for automatically maintaining a comprehensive, non-redundant protein database and for creating a quarterly release of this resource. In addition, we present tools for translating similarity searches into many annotation namespaces, e.g. KEGG or NCBI's GenBank. CONCLUSIONS: The data and tools we present allow the creation of multiple result sets using a single computation, permitting computational results to be shared between groups for large sequence data sets.

Connecting genotype to phenotype in the era of high-throughput sequencing.

BACKGROUND: The development of next generation sequencing technology is rapidly changing the face of the genome annotation and analysis field. One of the primary uses for genome sequence data is to improve our understanding and prediction of phenotypes for microbes and microbial communities, but the technologies for predicting phenotypes must keep pace with the new sequences emerging. SCOPE OF REVIEW: This review presents an integrated view of the methods and technologies used in the inference of phenotypes for microbes and microbial communities based on genomic and metagenomic data. Given the breadth of this topic, we place special focus on the resources available within the SEED Project. We discuss the two steps involved in connecting genotype to phenotype: sequence annotation, and phenotype inference, and we highlight the challenges in each of these steps when dealing with both single genome and metagenome data. MAJOR CONCLUSIONS: This integrated view of the genotype-to-phenotype problem highlights the importance of a controlled ontology in the annotation of genomic data, as this benefits subsequent phenotype inference and metagenome annotation. We also note the importance of expanding the set of reference genomes to improve the annotation of all sequence data, and we highlight metagenome assembly as a potential new source for complete genomes. Finally, we find that phenotype inference, particularly from metabolic models, generates predictions that can be validated and reconciled to improve annotations. GENERAL SIGNIFICANCE: This review presents the first look at the challenges and opportunities associated with the inference of phenotype from genotype during the next generation sequencing revolution. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Adaptive evolution of Toll-like receptor 5 in domesticated mammals.

BACKGROUND: Previous studies have proposed that mammalian toll like receptors (TLRs) have evolved under diversifying selection due to their role in pathogen detection. To determine if this is the case, we examined the extent of adaptive evolution in the TLR5 gene in both individual species and defined clades of the mammalia. RESULTS: In support of previous studies, we find evidence of adaptive evolution of mammalian TLR5. However, we also show that TLR5 genes of domestic livestock have a concentration of single nucleotide polymorphisms suggesting a specific signature of adaptation. Using codon models of evolution we have identified a concentration of rapidly evolving codons within the TLR5 extracellular domain a site of interaction between host and the bacterial surface protein flagellin. CONCLUSIONS: The results suggest that interactions between pathogen and host may be driving adaptive change in TLR5 by competition between species. In support of this, we have identified single nucleotide polymorphisms (SNP) in sheep and cattle TLR5 genes that are co-localised and co-incident with the predicted adaptive codons suggesting that adaptation in this region of the TLR5 gene is on-going in domestic species.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.